Measuring anion binding at biomembrane interfaces

The quantification of anion binding by molecular receptors within lipid bilayers remains challenging. Here we measure anion binding in lipid bilayers by creating a fluorescent macrocycle featuring a strong sulfate affinity. We find the determinants of anion binding in lipid bilayers to be different from those expected that govern anion binding in solution. Charge-dense anions H2PO4– and Cl– that prevail in dimethyl sulfoxide fail to bind to the macrocycle in lipids. In stark contrast, ClO4– and I– that hardly bind in dimethyl sulfoxide show surprisingly significant affinities for the macrocycle in lipids. We reveal a lipid bilayer anion binding principle that depends on anion polarisability and bilayer penetration depth of complexes leading to unexpected advantages of charge-diffuse anions. These insights enhance our understanding of how biological systems select anions and guide the design of functional molecular systems operating at biomembrane interfaces.

Na2SO4 (24 g) and NaHCO3 (0.15 g, 1.8 mmol) were dissolved in H2O (80 mL) at 30 °C. To this aqueous solution was added an EtOAc solution (40 mL) of 1,8-diaminocarbazole 1 (79 mg, 0.4 mmol) to form a biphasic mixture. Under vigorous stirring at 30 °C, triphosgene (83 mg, 0.28 mmol) was added to the biphasic mixture, and the stirring continued for 3 h at 30 °C. Afterwards, the organic phase which contained precipitates was separated from the aqueous phase using a separation funnel and carefully loaded into a prep-TLC plate (Analtech 02013) with frequent drying under a stream of N2 gas. The plate was initially run with MeCN for two hours to push the amine starting material to the top of plate, and then dried in a fumehood overnight. Triethylamine (TEA) was then pipetted into the baseline, and the prep-TLC was run with 9:1 (v:v) MeCN-TEA which led to separation of macrocycle 1 as a brown band from the baseline. The TEA treatment and the run with 9:1 (v:v) MeCN-TEA were repeated to isolate more macrocycle 1 from the baseline. After the plate was dry, the product band was scrapped and then treated with DMF (15 mL) to retrieve the product. The silica gel was removed by filtration and the DMF solution was evaporated under a stream of N2 gas overnight. The residue was triturated with EtOAc (2 mL) and dried under vacuum to give the 1-SO4 2complex as a grey powder (10 mg, 6%). The product contained 3-4 equivalents of TEA (Supplementary Figure 1), which could not be removed by vacuum drying or EtOAc trituration suggesting their strong interactions in the solid-state. Although Na2SO4 was used as the template, the Na + ions exchanged with metal ion impurities in the silica gel during prep-TLC separation. Therefore, the cationic component of the 1-SO4 2complex was a mixture of metal ions, determined to be (in mol%) 75% Zn, 12% Na, 7% Ca, 2% Mn, 2% Fe, 1% Al and 1% Mg by ICP-MS. 1 H NMR (400 MHz, DMSO-d6) δ 11.59 (s, 3H, carbazole-NH), 9.50 (s, 6H, urea-NH), 8.37 (d, J = 7.7 Hz, 6H, ArH), 7.76 (d, J = 7.7 Hz, 6H, ArH), 7.19 (t, J = 7.8 Hz, 6H, ArH), 2.98 (d, J = 6.5 Hz, 22H, TEA-CH2), 1.12 (t, J = 7.2 Hz, 33H, TEA-CH3). 13  S2. X-ray crystallography Single crystals of the 1-SO4 2complex (CCDC 2128483) was obtained by slow vapour diffusion of Et2O into an MeCN solution of 1-SO4 2-. A suitable crystal was selected and mounted with paratone on a MiTeGen Micromount. Data collection was performed on a Bruker APEX-II CCD diffractometer at 100(2) K. The highly disordered solvent molecules were removed using PLATON SQUEEZE. Using Olex2 2 , the structure was solved with the olex2.solve 3 structure solution program using Charge Flipping and refined with the SHELXL 4 refinement package using Least Squares minimisation.

S3. Computational modelling
Complexes of 1 with ClO4 -, NO3 -, 5 I -,Brand Cland were optimised at the B3LYP/6-31G * level of theory, using Spartan'14. As shown in Supplementary Figure 4, NO3fits perfectly into the macrocyclic cavity forming strong hydrogen bonds with all NH donors of 1 (b), leading to a perfectly flat and D3hsymmetric macrocycle, consistent with Mooibroek et al. 5 Although ClO4also fits well (a), the macrocycle is slightly buckled and one of the oxygen atoms of ClO4does not interact with the macrocycle. The three halide ions are too small for strong hydrogen bonding interactions with the macrocycle (c-e) as the NH donors only slightly (c, d) or hardly (e) overlap with the van der Waals sphere of the central halide ion.   1 H NMR titrations of free macrocycle 1 with TBA + salts of anions were performed in DMSO-d6/0.5% H2O. To obtain free macrocycle 1, an EtOAc solution of 1-Na2SO4 obtained after the reaction (see Section S1) was filtered to remove insoluble by-products and then the Na2SO4 was removed by extraction with Milli-Q water (40 mL). The EtOAc phase was evaporated at room temperature, and then the residue (free macrocycle 1) was immediately dissolved in DMSO-d6 (0.5 mL) and stored in a freezer for use in 1 H NMR titrations. Storage as a DMSO solution is necessary because free macrocycle 1 is unstable either as an EtOAc solution or in the solid state, but gains stability in DMSO likely due to the strong hydrogen bond accepting ability of DMSO. Because of partial degradation of the macrocycle in EtOAc upon Na2SO4 removal, the free macrocycle was 80-90% pure. This does not affect the validity of 1 H NMR binding studies because the signals from 1 and from impurities are well separated. The concentration of 1 in the DMSO-d6 stock solution was unknown, so this was determined by 1 H NMR integration against TBACl (as an internal reference) added to a 50-fold diluted DMSO-d6 solution of 1.

Supplementary
For each titration experiment, a 0.3 mM solution of free macrocycle 1 was prepared in DMSO-d6 with 0.5% (vol%) of added H2O, and then 1 H NMR spectra in the absence and presence of increasing concentrations of TBA + salts of anions were acquired. The chemical shifts of the carbazole NH, urea NHs and one aromatic CH that was the most sensitive to anion addition among the three CHs were plotted against the anion concentration. In the case of NO3 -, the aromatic CH was not used for analysis because it converged and then swapped positions with another aromatic CH during the titration. Data fitting was performed using online software Bindfit 6 to calculate the binding constants.
It should be noted that we found the commercial DMSO-d6 to contain ~15 μM of SO4 2impurities, and therefore signals of 1-SO4 2complex (in slow exchange with other species) were always observed except with excess of HPO4 2which could fully displace SO4 2from 1 (Supplementary Figure 14 top spectrum).
Selection of the binding model (1:1 or 1:2) was based on the observed shape of the binding isotherm, as well as the knowledge of the charge-density of the anions and their extent of size/shape-matching with the macrocycle. In the case of NO3 -, the binding isotherm is characteristic of a 1:1 equilibrium with saturation observed at high anion concentrations (Supplementary Figure 10). This, in combination with the perfect size/shape-matching of NO3with the macrocycle (Supplementary Figure 4b) and the weak charge-density of NO3renders the binding of a second NO3 -(which does not benefit from macrocyclic pre-organisation and multivalency) unlikely, justifying the use of the 1:1 binding model. By contrast, for more charge-dense and less structurally fitting Cland Branions ( Supplementary  Figures 4c, 4e), some of the 1 H signals continued shifting at high anion concentrations after initially approaching saturation (Supplementary Figures 6, 8), indicating a stepwise 1:2 equilibria. This is plausible because the affinity of the first Cl -/Bris compromised by the poor structural fitting, whereas the binding of second anion is more pronounced than in the case of NO3due to higher charge densities of Cland Br -. For I -, the 1:1 model was used as the low affinity renders the binding of a second Iunlikely to be significant at the tested concentration range.   Slow exchange signals were observed upon titration of the free macrocycle 1 with TBA2SO4 (Fig. 2a).
Integration of the TBA + and the 1-SO4 2signals confirms the 1:1 stoichiometry. Free macrocycle 1 was completely converted to the SO4 2complex at 1.0 equiv. of TBA2SO4, indicating a very strong SO4 2affinity.
To determine the SO4 2affinity of 1, we used a BaSO4 precipitation method taking advantage of a recently reported Ksp value of BaSO4 in DMSO. 8 To two solutions of 1-SO4 2-(100 μM) in DMSO-d6/0.5% H2O was added BaCl2 (solids) to 50 mM and 100 mM, respectively. The solutions were sonicated until the BaCl2 dissolved and then incubated for a week for Ba 2+ to extract SO4 2from 1 and form BaSO4. The 1 H NMR of the two solutions were acquired, both of which showed separate sets of signals from 1-SO4 2and 1-Cl -(here 1-Clrefers to a fast-exchanged mixture of 1, 1-1Cland 1-2Cl -), respectively (Supplementary Figure 15). By integration of the urea NH, we calculated the [1- , we calculated the Ba 2+ activity coefficients to be 0.027 and 0.059 for BaCl2 at 50 mM and 100 mM, respectively. Finally, the binding constant of 1 for SO4 2was calculated using the following equation:
The derivation of Supplementary Equation 1 is shown as follows: S14 For SO4 2binding to macrocycle 1:

Supplementary Equation 2
For the BaSO4 precipitation equilibrium:

Supplementary Equation 6
where Titrations of 1 with SO4 2and H2PO4were performed in C12E8 micelles. 12.5 μL of a DMSO solution of 1-SO4 2-(10 μM) was added to 2.5 mL of a micellar solution of C12E8 (2 mM) to a final macrocycle concentration of 50 nM. Because of the low concentration used, the 1-SO4 2complex completely dissociated into free macrocycle 1 (see Supplementary Figure 28 for evidence of its stability under the C12E8 micellar conditions). The solution in a 4 mL quartz cuvette was stirred and thermostated at 25 °C. Fluorescence spectra of 1 (λex = 265 nm) before and after adding increasing concentrations of Na2SO4 or NaH2PO4 were recorded. For SO4 2-, the fluorescence intensity at 357 (corrected against dilution) was plotted against the concentration of SO4 2and the data was fitted to a 1:1 binding models using OriginPro. For H2PO4 -, the fluorescence intensity values from 350 to 380 nm were globally fitted to the 1:2 (host:guest) model with dilution correction in BindFit. Note that here the use of the 1:2 model was to account for the slight dynamic fluorescence quenching observed with high concentrations of H2PO4and the actual formation of the 1:2 complex should be negligible under the competitive aqueous conditions.
The Na2SO4 titration of 1 was also performed using 9:1 C12E8/POPC and 8:2 C12E8/POPC (mol:mol) mixed micelles to estimate competitive phosphate headgroup binding, following the abovementioned procedure except that POPC was used with the total C12E8 + POPC concentration kept constant at 2 mM. Note that the presence of 10% or 20% of POPC did not appear to impact the self-assembly of C12E8 micelles, as DLS measurement showed an identical hydrodynamic radius of 3.5 ± 0.1 nm for 100% C12E8  and ClO4 -(pink) induced fluorescence responses, which in part originated from a dynamic quenching mechanism, so the binding constants calculated from the fluorescence titration data would be incorrect in those cases. To determine the affinities of 1 for anions other than SO4 2and H2PO4 -, we performed the Na2SO4 titration in the presence of 200 mM of the tested anion as a competitive binder. Instead of collecting the full fluorescence spectra, we only recorded the fluorescence intensities at 357 nm and 365 nm in competition titrations to minimise photobleaching. The binding constant of the competitive binder could be calculated by a competition binding scheme using the SO4 2affinities in the absence and presence of the competitive binder. However, surface potential effects need to be corrected because anions can adsorb to non-ionic micelles 11 resulting in a negative surface potential which depletes SO4 2ions from the solution by a Boltzmann factor.
To correct for surface potential effects, we determined zeta potential of C12E8 (2 mM) micelles in the presence of 200 mM of salts by electrophoretic mobility measurements at 25 °C, using DTS1070 folded capillary cells. Note that in the case of NaI, it is necessary to reduce the applied voltage to minimise the electrolysis of I -, which produces more lipophilic I3and enhances the electrophoretic mobility of micelles. We also determined the size of the micelles in the presence of salts at 200 mM and found the hydrodynamic diameter to be 3.7 ± 0.3 nm (similar to the value without salts) regardless of the identity of the salt. The zeta potential (ζ) was calculated using Henry's equation: where μe is the electrophoretic mobility, η is the viscosity of the water, 0 is the vacuum permittivity, is the relative permittivity of water, and f(κa) is Henry's function (calculated to be 1.16 using the Malvern Zetasizer software for C12E8 micelles with a hydrodynamic radius of 3.7 nm).

S19
The zeta potentials were then converted to surface potentials (Φ0) using the following relationship from the Gouy-Chapman model: 12 where Φx is the zeta potential (ζ), Z = -1, F is the Faraday constant, R is the gas constant, κ is the inverse of Debye length, and x is the distance of the shear plane from the surface (assumed to be 0.6 Å as determined by Yang et al. for similarly-sized micelles at I = 0.2 M 13 ).
The zeta potential and surface potential values are summarised in Supplementary Table 2.

S6.1 Evidence of anion binding
We found that good-to-reasonable quality 1 H NMR spectra of anion complexes of macrocycle 1 (at μM concentrations) in C12E8 micelles could be obtained using a 600 MHz NMR spectrometer equipped with a cryoprobe, providing direct evidence of anion binding in C12E8 micelles. For solution preparation, 5 μL of a DMSO-d6 solution of 1-SO4 2-(0.5 mM) was added to a 495 μL solution of C12E8 (50 mM) in 9:1 (v:v) H2O/D2O, to a final macrocycle concentration of 5.0 μM. Solutions containing 2 mM of Na2SO4 or 200 mM of NaH2PO4, NaCl, NaBr, NaNO3, NaI, or NaClO4 were also prepared. The zgesgppe pulse programme was used to suppress the water signal. The spectra were referenced to the DMSO-d5 signal (set as 2.71 ppm). 1 Figure 28 for the carbazole NH signal of 1-SO4 2at 11.7 ppm. Note that some signals are invisible due to broadening or overlapping with signals from C12E8. The spectra for NaBr and NaClO4 were scaled up due to low signal intensities, likely indicating Brand ClO4enhancing aggregation of 1 under the current conditions. In fluorescence titrations, we used 100-fold diluted macrocycle 1 to minimise aggregation of 1. The remaining signals are from C12E8 or impurities in the solvent/C12E8. . The vesicles were prepared following the procedure in S8.1, except that 200 mM NaCl solution instead of pure water was used to hydrate the lipids. Fluorescence titration of 1 (50 nM) with Na2SO4 was performed following the procedure in S8.1, except that the vesicles were suspended in an isotonic NaX + NaCl (total concentration 200 mM, Xis a competing anion) solution and that only fluorescence intensities at 358 nm and 349 nm were collected to minimise photobleaching. For each competing anion, we performed competitive Na2SO4 titrations at two concentrations.
We corrected surface potential effects by determining zeta potential of POPC (2 mM) vesicles in the presence of salts via electrophoretic mobility measurements at 25 °C, using DTS1070 folded capillary cells. The vesicles after extrusion were degassed for 8 min under vacuum. 14 Note that in the case of NaI, it was necessary to reduce the applied voltage to minimise the electrolysis of I -, which produces more lipophilic I3and enhances the electrophoretic mobility of vesicles. We also determined the size of the vesicles in the presence of salts at 200 mM and found the hydrodynamic radii to be consistently 65 ± 5 nm regardless of the identity of the salt. The zeta potential (ζ) was calculated by Henry's equation (Supplementary Equation 7) using a f(κa) value of 1.45 calculated by the Malvern Zetasizer software. The surface potential was then calculated from the zeta potential using Supplementary Equation 8, assuming the shear plane to be 2 Å 15 from the surface (Supplementary Table 3).

S28
Supplementary Note that vesicles in 200 mM NaH2PO4 exhibit a positive zeta potential because of the preferential adsorption of Na + cations 14 over H2PO4anions to vesicles.
For competition titrations with mixed salts (e.g., 50 mM of NaNO3 and 150 mM of NaCl to keep the ionic strength at 0.2 M), we calculated the Φ0 values assuming linear relationship between Φ0 and the anion fraction. 16 For Iand ClO4 -, however, the relationship is not linear due to high affinities of these anions for the lipids (Table 1). In these cases, we experimentally determined the ζ and calculated the Φ0 values under mixed salt conditions (Supplementary Table 4).

Supplementary Table 4
Zeta potential (ζ) and calculated surface potential (Φ0) of POPC vesicles (2 mM) in the presence of mixed salts. The values are shown as average ± SD from at least three measurements.

S8. Control fluorescence studies
To verify that the formation of the 1-SO4 2complex occurs within C12E8 micelles or POPC membrane, instead of in the bulk solution, we recorded the fluorescence spectra of 1 in the absence and presence of Na2SO4 in pure water. 12.5 μL of a DMSO solution of 1-SO4 2-(10 μM) was added to 2.5 mL of water or a Na2SO4 (20 mM) solution in water, stirred and thermostated at 25 °C in a 4 mL quartz cuvette. Supplementary Figure 39 shows that neither the free macrocycle 1 or its SO4 2complex is fluorescent in pure water due to insolubility, confirming that SO4 2binding occurs in the micelle/membrane phase instead of in the bulk solution.  Figure 41) is likely due to slightly increased solubility of 3 because the shape of the fluorescence spectrum with SO4 2is identical to that of the free receptor. Therefore, the strong fluorescence response to SO4 2is unique to macrocycle 1. where zcf is the distance of macrocycle 1 from the bilayer centre, F1 is the fluorescence intensity (we used the intensity at 360 nm) of macrocycle 1 in the presence of quencher 1 (the shallow quencher TEMPO PC), F2 is the fluorescence intensity (we used the intensity at 360 nm) of macrocycle 1 in the presence of quencher 2 (the deeper quencher 5-doxyl PC), Lc1 is the distance of quencher 1 from the bilayer centre, L21 is the distance between quenchers 1 and 2, C is the quencher concentration in S35 molecules/Å (= mole fraction of spin-labelled lipids/70 Å 2 ). The distances of the nitroxide quencher from bilayer centre was previously estimated to be 19.   S37 S10. Transmembrane anion transport S10.1 HPTS assay

Supplementary
We have decided to use the salt-pulse assay 19 for quantitative determination of anion transport rates and selectivity, because the more commonly used base-pulse assay is compromised by potential aniondependent transporter partitioning 20 and surface charge effects due to membrane adsorption of chaotropic anions. 16,21,22 These effects are more significant in the base-pulse assay (anions used at 100 mM) than in the salt-pulse assay (anion concentration can be reduced to 20 mM or lower). In this assay, the anion transporter facilitates anion − and H + influx driven by the anion concentration gradient, leading to acidification of the vesicle interior as monitored by the ratiometric fluorescence response of the intravesicular pH indicator HPTS. We used sodium gluconate (NaGluc) as the main osmolyte because Gluc − is a large, hydrophilic anion that cannot be transported across the membrane. To convert the raw HPTS fluorescence data into transport rates in number of anions per second per carrier, we first performed a calibration of the HPTS response against pH for NaGluc in /NaGluc out vesicles using a previously described procedure. 20 Here the relationship between the pH (y) and I460/I403 of HPTS ( The initial rate (k) is then calculated using the following equation: Finally, the initial rate of H + influx in mM s −1 using a transporter at a given mol% loading is converted to anions s −1 carrier −1 , using the following equation obtained after taking literature values of the vesicle volume and the number of POPC molecules per vesicle for 200 nm (diameter) POPC LUVs, 24 noting that the influx of each H + is accompanied by an anion (for monovalent anions): transport rate in anions s −1 carrier −1 = 0.67 × transport rate in mM s −1 carrier loading in mol% with respect to lipid

Supplementary Equation 16
For SO4 2− , the value calculated by Supplementary Equation 16 was divided by 2.
Note that: (1) Here without correction of the carrier deliverability, 25 the carrier refers to all carriers added into the solution instead of active carriers that partitioned in the membrane; (2) The rate depends on the ion concentration used and usually increases linearly with increasing ion concentration until the carriers are saturated by ion binding. 26

S10.2 Osmotic response assay
The HPTS assay has established macrocycle 1 as an H + /X − symporter. To examine whether 1 can also function as an X − uniporter, we performed an osmotic response assay to monitor NO3 − transport in vesicles (Supplementary Figure 49). In this assay, under the conditions shown in Supplementary Figure  49a, the anion transporter needs to facilitate NO3 − uniport to couple to K + uniport by valinomycin (Vln) to give overall KNO3 efflux. In Supplementary Figure 49b, the anion transporter needs to facilitate H + /NO3 − symport to couple to K + /H + antiport facilitated by monensin to give overall KNO3 efflux. 27 KNO3 efflux causes osmotic shrinkage and an increase of light scattering intensity of vesicles as monitored using a fluorometer. 28 Supplementary Figure 49 Schematic representation of the osmotic response assay to investigate NO3 − uniport (a, with valinomycin, Vln) and H + /NO3 − symport (b, with monensin, Mon) facilitated by 1.

S40
The assay was conducted using POPC LUVs (mean diameter 400 nm) loaded with KNO3 (300 mM) and suspended in KGluc (300 mM). Both the internal and external solutions were buffered at pH 7.0 with 10 mM of HEPES. The POPC LUVs were prepared as follows. 2 mL of a chloroform solution of POPC (1 g in 35 mL) was evaporated in a round-bottom flask and the lipid film formed was dried under vacuum for at least 6 h. Then, the lipid film was hydrated by vortexing with a 2 mL internal solution of KNO3 (300 mM) buffered at pH 7.0. The lipid suspension was subjected to nine freeze/thaw cycles and then extruded 21 times through a 400 nm polycarbonate membrane. The concentration of POPC (~40 mM) was calculated based on the weight difference of the round bottom flask before POPC addition and after drying.
For each measurement, the vesicles were diluted to a final POPC concentration of 0.2 mM using an external KGluc (300 mM) solution buffered at pH 7.0. The sample in a 4 mL disposable polystyrene cuvette was stirred and thermostated at 25 °C. The 90° light scattering intensity of vesicles (λex = 600 nm, λem = 600 nm) was recorded by a fluorometer. 10 μL of a DMSO solution of 1-SO4 2-(0.5 mM) was added (final concentration of 1 was 2 μM or 1 mol% with respect to lipids), followed by valinomycin (0.1 μM, 0.05 mol%) or monensin (1 μM, 0.5 mol%) at t = 0 s. The transport kinetics was monitored for at least 1 h. At the end of the experiment, N,N′-bis [3,5-bis(trifluoromethyl)phenyl]-thiourea (4 μM, 2 mol%, 1 μM of monensin was also added if no cationophore was present previously) was added to complete the salt transport and normalise the light scattering intensity to 100% salt efflux.
Macrocycle 1 induced significant NO3 − transport in the presence of monensin, but did not increase NO3 − transport relative to the valinomycin baseline (Supplementary Figure 50), confirming that 1 functions as a H + /NO3 − symporter but not as a NO3 − uniporter. Note that salt efflux could be observed with valinomycin alone, which is attributed to facilitated transport via a valinomycin-KNO3 ion pair complex. 29  [3,5-bis(trifluoromethyl)phenyl]-thiourea (2 mol%, 0.5 mol% of monensin was also added if no cationophore was present previously) was added to complete salt transport and normalise the light scattering intensity to 100% salt efflux. Error bars represent SD from two experiments.